The long-term objectives of this project are to define the mechanism of vitamin B12 uptake in Escherichia coli and to demonstrate the location and interaction of all components of this transport system. This uptake system shares the unusual property with iron uptake systems that they employ substrate-specific receptor proteins in the outer membrane as essential components of the high-affinity uptake process. Receptor function is also dependent on the tonB and exbB genes, and separate loci appear to be involved in transport of the substrate across the cytoplasmic membrane. During the current period, we have cloned and sequenced all three of the loci directly involved in vitamin B12 uptake. We plan to use this information to guide in vitro mutagenesis of the cloned btuB gene and protein labeling and antibody probing of the B12 receptor with the goal of defining the various functional, substrate-binding domains on the receptor. These studies will also initiate development of a model for the topology of the receptor in the outer membrane. Additional studies will be directed to the definition of the regulation of expression of btuB, which is repressed by growth in B12 and also affected by temperature and osmolarity. For this study, btuB-lac operon fusions will allow isolation of regulatory mutants with altered expression. The other branch of this project relates to the possible interaction of the tonB product with the receptor. Studies of the cellular location of TonB and of its binding in a receptor density-dependent manner to the outer membrane will make use of radio-labeled TonB protein produced in maxicell systems. Attempts to obtain quantities of TonB and antibodies against it will make use of high-level protein expression vectors and tonB-lac hybrid proteins constructed in vitro. Selection protocols will be developed to identify genetic loci which affect TonB function and to alter domains on the receptors at which TonB might interact. Finally, a speculative side-project will explore the possibility that the btuB-tonB system might mediate entry of B12 into the cytoplasm without involvement of the btuC function. The relation of this process to the uptake of colicins, which utilize the B12 receptor will be explored, initially by examination of the requirement for btuC function for B12 uptake in strains tolerant to the colicins. It is hoped that this project will lend some insight into this unusual transport process and into binding sites.